Self-(in)compatibility of the almonds P. dulcis and P. webbii: detection and cloning of 'wild-type Sf ' and new self-compatibility alleles encoding inactive S-RNases

Prunus dulcis, the almond, is a predominantly self-incompatible (SI) species with a gametophytic self-incompatibility system mediated by S-RNases. The economically important allele S _f , which results in self-compatibility in P. dulcis, is said to have arisen by introgression from Prunus webbii in the Italian region of Apulia. We investigated the range of self-(in)compatibility alleles in Apulian material of the two species. About 23 cultivars of P. dulcis (14 self-compatible (SC) and nine SI) and 33 accessions of P. webbii (16 SC, two SI and 15 initially of unknown status), all from Apulia, were analysed using PCR of genomic DNA to amplify S-RNase alleles and, in most cases, IEF and staining of stylar protein extracts to detect S-RNase activity. Some amplification products were cloned and sequenced. The allele S _f was present in nearly all the SC cultivars of P. dulcis but, surprisingly, was absent from nearly all SC accessions of P. webbii. And of particular interest was the presence in many SI cultivars of P. dulcis of a new active allele, labelled S ₃₀ , the sequence of which showed it to be the wild-type of S _f so that S _f can be regarded as a stylar part mutant S ₃₀ °. These findings indicate S _f may have arisen within P. dulcis, by mutation. One SC cultivar of P. dulcis, ‘Patalina’, had a new self-compatibility allele lacking RNase activity, S _n5 , which could be useful in breeding programmes. In the accessions of P. webbii, some of which were known to be SC, three new alleles were found which lacked RNase activity but had normal DNA sequences.     

Cell reabsorption in the Euphorbia dulcis endosperm

Euphorbia dulcis endosperm is the site of controlled long lasting endocellular lysis involving segregation and autophagy of portions of the cytoplasm within the endoplasmic reticulum membrane. The lysis products may constitute a food source for the benefit of the early developing embryo.     

Triterpenes from periandra dulcis roots

Two new triterpenes from the root of Periandra dulcis were found to be 3,25-dioxoolean-12(13)-en-30-oic acid and 3,25-dioxoolean-18(19)-en-30-oic acid.     

Chlorophyll fluorescence performance of sweet almond [Prunus dulcis (Miller) D. Webb] in response to salinity stress induced by NaCl

One-year old sweet almond (Prunus dulcis) seedlings were submitted to four levels of salt stress induced by NaCl, namely 0.3, 0.5, 0.7, and 1.0 S m⁻¹. Effects of salt stress on a range of chlorophyll (Chl) fluorescence parameters (Chl FPs) and Chl contents were investigated in order to establish an eco-physiological characterization of P. dulcis to salinity. Salt stress promoted an increase in F₀, F_s, and F₀/F_m and a decrease in F_m, F′_m, F_v/F_m, q_P, ΔF/F′_m, F_v/F₀, and UQF_(rel), in almost all Chl fluorescence yields (FY) and FPs due to its adverse effect on activity of photosystem 2. No significant changes were observed for quenchings q_N, NPQ, and q_N(rel). The contents of Chl a and b and their ratio were also significantly reduced at increased salt stress. In general, adverse salinity effects became significant when the electric conductivity of the nutrient solution (EC_n) exceeded 0.3 S m⁻¹. The most sensitive salt stress indicators were F_v/F₀ and Chl a content, and they are thus best used for early salt detection in P. dulcis. Monitoring of a simple Chl FY, such as F₀, also gave a good indication of induced salt stress due to the significant correlations observed between the different Chl FYs and FPs. Even essential Chl FYs, like F₀, F_m, F′_m, and F_s, and mutually independent Chl FPs, like F_v/F₀ and q_P, were strongly correlated with each other.     

Triterpenoids of Scoparia dulcis

The triterpenoids of Scoparia dulcis were identified as friedelin, glutinol, α-amyrin, betulinic acid, ifflaionic acid and dulcioic acid.     

New labdane diterpenes from the aerial parts of Scoparia dulcis L.

Three new labdane-derived diterpenes, dulcinodal (1), dulcinodiol (2), and scopadiol decanoate (3) were isolated from the aerial parts of Scoparia dulcis. The structures were determined by extensive NMR studies and comparison of their spectral data with related compounds.     

Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants

Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (β-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed to selection 20 days after cocultivation. From 1419 original leaves, four shoots (A, B, C and D) were obtained that showed amplification of the predicted DNA fragments by polymerase chain reaction (PCR). After micropropagation of these shoots, only those cloned from shoot D gave consistently positive results in histochemical GUS detection and PCR amplification. Southern blot hybridisation confirmed stable transgene integration in clone D, which was also negative in PCR amplification of an Agrobacterium gene. Additional molecular analysis suggested that the remaining three shoots (A, B and C) were chimeric. Received: 28 March 1998 / Revision received: 18 April 1998 / Accepted: 12 May 1998     

Improved in vitro rooting of Prunus dulcis Mill. cultivars

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47–100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm⁻³ gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.     

A re-examination of the cloacal sacs and gland of the blind snake,Leptotyphlops dulcis (Reptilia: Leptotyphlopidae)

The cloacal sacs of Leptotyphlops dulcis are nonglandular, posterior evaginations of the cloaca. The median cloacal gland is tubuloalveolar. Similar unpaired cloacal glands as well as paired sacs are noted in certain colubrid snakes. Terminology applied to these cloacal derivatives is discussed, and a standardization of names is provided.     

A new subspecies, Polynemus melanochir dulcis, from Tonle Sap Lake, Cambodia, and redescription of P. m. melanochir Valenciennes in Cuvier and Valenciennes, 1831 with designation of a neotype

 A poorly known threadfin, Polynemus melanochir Valenciennes in Cuvier and Valenciennes, 1831, is redescribed (as P. melanochir melanochir) on the basis of a newly designated neotype and a large number of specimens. Polynemus melanopus Sauvage, 1881 and 3 species commonly regarded as valid, Galeoides microps Steindachner, 1869, P. borneensis Bleeker, 1857, and Trichidion hilleri Fowler, 1905, are all considered junior synonyms of P. m. melanochir. In addition, a new subspecies, P. melanochir dulcis, is described on the basis of 3 specimens. Polynemus m. dulcis differs from P. m. melanochir in having a greater snout length [7% of SL vs. mean 6% (range 5–6%) of SL in the latter] and shorter upper caudal-fin lobe length [35% (34–35%) of SL vs. 39% (35–44%) of SL]. The former is currently known only from Tonle Sap Lake, Cambodia, whereas the latter is known from the Mekong River (Cambodia and southern Vietnam) and Kalimantan (=Borneo; Malaysia and Indonesia). Received: September 27, 2001 / Revised: December 20, 2001 / Accepted: December 29, 2001     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1 mg l⁻¹ indole-3-butyric acid and 15 mg l⁻¹ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for β-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation system. It took only 2–3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.     

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Summary Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15–60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

In vitro rooting of almond (Prunus dulcis Mill.)

Summary Shoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.     

Inhibition of α-Glycosidase by Lippia dulcis Trevir. (Verbenaceae) Preparations,Quantification of Verbascoside,and Study of Its Molecular Docking

This study aimed to quantify verbascoside (VEB), perform molecular docking studies of VEB with the α-glucosidase (GL) of Bacillus stearothermophilus, and evaluate the inhibition of the enzyme by L. dulcis preparations. The substrate concentration and presence of reduced glutathione were evaluated for their effect on the in vitro inhibition of the GL enzyme. Assays were also performed in the presence and absence of simulated gastric fluid. The antidiabetic fractions 2 and 3 were the most inhibited GL, but their activity were significantly decreased in the presence of gastric fluid. Chromatographic analyses confirmed the predominant presence of VEB in the samples. The samples had VEB concentrations between 49.9 and 243.5 mg/g. Simulation of the molecular docking of VEB were consistent with its GL-inhibitory activity. It can conclude that the crude ethanol extract and fractions show inhibitory activity against the GL enzyme.     

The RNase PD2 gene of almond (Prunus dulcis) represents an evolutionarily distinct class of S-like RNase genes

A cDNA for an S-like RNase (RNase PD2) has been isolated from a pistil cDNA library of Prunus dulcis cv. Ferragnés. The cDNA encodes an acidic protein of 226 amino acid residues with a molecular weight of 25 kDa. A potential N-glycosylation site is present at the N-terminus in RNase PD2. A signal peptide of 23 amino acid residues and a transmembrane domain are predicted. The two active-site histidines present in enzymes of the T2/S RNase superfamily were detected in RNase PD2. Its amino acid sequence shows 71.2% similarity to RNS1 of Arabidopsis and RNase T2 of chickpea, respectively. Northern blotting and RT-PCR analyses indicate that PD2 is expressed predominantly in petals, pistils of open flowers and leaves of the almond tree. Analyses of shoots cultured in vitro suggested that the expression of RNase PD2 is associated with phosphate starvation. Southern analysis detected two sequences related to RNase PD2 in the P. dulcis genome. RFLP analysis showed that S-like RNase genes are polymorphic in different almond cultivars. The PD2 gene sequence was amplified by PCR and two introns were shown to interrupt the coding region. Based on sequence analysis, we have defined three classes of S-like RNase genes, with the PD2 RNase gene representing a distinct class. The significance of the structural divergence of S-like RNase genes is further discussed. Received: 24 January 2000 / Accepted: 17 March 2000     

Association of Elm Yellows Subgroup 16SrV‐B Phytoplasma with a Disease of Hovenia dulcis

Japanese raisin (Hovenia dulcis) trees with typical phytoplasma‐like symptoms were observed for the first time in South Korea. The disease, named Japanese raisin witches’ broom, is progressively destructive. The cause of the graft‐transmissible disease was confirmed by electron microscopy and molecular studies. The 16S rDNA sequence analysis showed that the phytoplasma was closely related to the elm yellows (EY) group, ribosomal subgroup 16SrV‐B. The 16S‐23S rDNA intergenic spacer region, fragment of rp operon and secY gene sequences had 96–99% similarity with members of EY phytoplasma. Based on the sequence analyses and phylogenetic studies, it was confirmed that the phytoplasma infecting Japanese raisin trees in Korea belongs to the EY group.     

Contributo All'Embriologia Delle Plumbaginaceae (con 103 figg. nel testo)

Riassunto

L'A. stabilisce che in Plumbago capensis Thunb., accanto allo sviluppo secondo lo schema del tipo Plumbago, si riscontra, con bassissima frequenza, un nuovo tipo di sviluppo di gametofiti 7-nucleati bipolarizzati con oosfera di natura sporiale.

Stabilisce inoltre che in Statice Limonium L. lo sviluppo del gametofito femminile avviene esclusivamente secondo il tipo Euphorbia dulcis, mentre in Armeria vulgaris W. var. maritima (Miller) Willd. esiste associazione dei due tipi di sviluppo Adoxa ed Euphorbia dulcis nel rapporto approssimativo del 3%.

Mette in evidenza che la triploidia dei nuclei calazali, caratteristica dei gametofiti di tipo Euphorbia dulcis, può determinarsi secondo due distinte modalità : o per coalescenza dei tre fusi calazali durante la terza divisione dello sviluppo (Statice Limonium), o per un processo di vera e propria cariogamia dei tre nuclei sporiali quiescenti durante lo stadio di polarizzazione 1+3 con conscguente formazione durante la storia dello sviluppo di uno stadio binucleato secondario (Armeria vulgaris var. maritima).

Riconferma infine per questa ultima pianta il numero aploide dei cromosomi n = 9.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Hypolipidemic phenanthraquinone derivatives from Heleocharis dulcis

Three new phenanthraquinones (1–3), together with seven known phenanthraquinone derivatives (4–10) were isolated from Heleocharis dulcis for the first time. Their structures were established on extensive NMR, MS, UV, IR experiments in conjunction with their references. Compounds (1–10) were evaluated for their hypolipidemic activities for the first time. Compounds 1, 3, 7, and 9 (10 μM) exhibited significant hypolipidemic activities by measuring the triglyceride content in HepG2 cells with simvastatin as positive control. The chemotaxonomic significance of compounds (1–10) was also discussed.